ARTIGO/ARTICLE
Revista da Sociedade Brasileira de Medicina Tropical 41(4):, jul-ago, 2008
Human serum antibody reactivity towards Paracoccidioides brasiliensis
antigens treated with sodium metaperiodate
Reatividade de anticorpos de soros humanos a antígenos de
Paracoccidioides brasiliensis tratados com meta-periodato
Ana Paula Ferreira1, Thadeu Côrrea1, Rosângela Cunha2, Marcos José Marques1,
Maria Angela Montesano1, Maria Aparecida Souza1 and Henrique Couto Teixeira1.
ABSTRACT
In this study, we evaluated the profile of anti-Paracoccidioides brasiliensis immunoglobulin isotypes in serum from patients with the acute and
chronic forms of paracoccidioidomycosis, using the whole Paracoccidioides brasiliensis antigen and the antigen treated with sodium metaperiodate.
All the immunoglobulin isotypes present in the serum from patients with the acute and chronic forms of paracoccidioidomycosis presented higher
reactivity towards the whole antigen than to the antigen treated with metaperiodate (P < 0.05). The reactivity of IgG and IgM to the antigen treated with
metaperiodate was greater in serum from patients with the acute form of the disease (P < 0.05), while IgA was more reactive in serum from patients
with the chronic form (P < 0.05). There was greater reactivity of IgG1 and IgG2 to the whole antigen and the antigen treated with metaperiodate in
the serum from patients with paracoccidioidomycosis than there was in serum from patients with other parasitic infections (P < 0.05). Furthermore,
IgG1 from patients with the acute form recognized the 19 kDa, 27 kDa and 31 kDa antigens in the western blot test. Thus, the results suggest that
modifications to the epitopes of Paracoccidioides brasiliensis antigens may help to improve the immunodiagnosis of paracoccidioidomycosis.
Key-words: Paracoccidioidomycosis. Antigens. Metaperiodate. Diagnosis.
����������
RESUMO
Neste trabalho, foi avaliado o perfil de isotipos de imunoglobulinas anti-Paracoccidioides brasiliensis em soros de pacientes com formas crônica
e aguda de paracoccidiodomicoses usando antígeno total e tratado com meta-periodato. Todos os tipos de imunoglobulinas presentes nos soros
de pacientes com formas aguda e crônica apresentaram alta reatividade ao antígeno total quando comparado ao tratado com meta-periodato (P <
0,05). Houve maior reatividade de IgG e IgM anti-antígeno tratado com meta-periodato em soros de pacientes com forma aguda da doença (P <
0,05), enquanto IgA foi mais reativa em soros da forma crônica (P < 0,05). Houve maior reatividade de IgG1 e IgG2 com antígeno total e tratado
com meta-periodato em soros de pacientes comparados aos com outras parasitoses (P < 0,05). Além disso, IgG1 de pacientes com a forma aguda
reconhecem antígenos de 19kDa, 27kDa e 31kDa por “western blot”. Assim, os resultados sugerem que alterações nos epitopos de antígenos de P.
brasiliensis podem auxiliar no aprimoramento do imunodiagnóstico da paracoccidioidomicose.
Palavras-chaves: Paracoccidiodomicose. Antígenos. Meta-periodato. Diagnóstico.
Paracoccidioidomycosis (PCM), a granulomatous disease
caused by the dimorphic fungus Paracoccidioides brasiliensis,
is the most prevalent systemic mycosis in Latin America4 5.
Symptomatic individuals show one of two main clinical forms of
the disease: the acute or subacute form, which is characterized
by rapid and progressive infection and largely affects the
mononuclear phagocyte system; and the chronic form, which
frequently shows a slow course of pulmonary disease with
mucocutaneous involvement.
The criteria for laboratory diagnosis of PCM rely on positive
microscopic identification of the causative agent in KOH
preparations or biopsy specimens, as well as on its isolation in
cultures5 12. In addition, diagnostic methods based on serological
tests have also been widely used to support identification of
microscopy-positive cases5 14 and represent an important tool for
follow-up of PCM patients6. However, serological tests normally
use crude antigenic preparations of P. brasiliensis, which may
be cross-reactive with tests for other mycotic diseases8. Serum
1. Laboratório de Imunologia, Dept° de Parasitologia, Microbiologia e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Juiz de Fora, MG. 2. Serviço de
Doenças Infecciosas e Parasitárias, Hospital Universitário, Universidade Federal de Juiz de Fora, MG.
Financial Support: CNPq, FAPEMIG and CAPES
Address to: Dra. Ana Paula Ferreira. Laboratório de Imunologia/ Dept° de Parasitologia, Microbiologia e Imunologia/ ICB/ UFJF. 36036-900 Juiz de Fora, MG.
Tel e fax: 55 32 2102-3214
e-mail: [email protected]
Recebido para publicação em 18/4/2008
Aceito em __/__/__
Ferreira AP et al
cross-reactivity of PCM with histoplasmosis and Jorge Lobo’s
disease has also been detected when 43-kDa glycoprotein, an
exocellular antigen obtained from a liquid culture medium of P.
brasiliensis, is used. Cross-reactivity towards gp43 seems to be
due to carbohydrate epitopes, since more than 85% of the specific
reactions of gp43 with serum antibodies from PCM patients have
involved protein epitopes17.
In this study, treatment with sodium metaperiodate was used
to disrupt the carbohydrate epitopes of P. brasiliensis filtrate
antigens and generate a purified protein antigenic preparation.
Whole P. brasiliensis soluble antigens (WAg) and sodium
metaperiodate-treated WAg (MPAg) were used to evaluate the
profile of serum immunoglobulin isotype (total IgG, IgM, IgA,
IgG1 and IgG2) reactivity towards P. brasiliensis in patients
with acute or chronic forms of PCM. Furthermore, we used the
western blot method to investigate serum IgG1 reactivity towards
fractionated P. brasiliensis antigens in serum from acute and
chronic PCM patients.
MATERIAL AND METHODS
Patients and controls. The study was conducted on serum
samples obtained from twenty-nine patients with active PCM
with either the acute form (n=15) or chronic form (n=14) of
the disease. All the patients had PCM, according to the clinical
diagnosis of symptoms that indicates presence of P. brasiliensis
infection, and according to positive cultures and immunodiffusion
tests for crude P. brasiliensis antigens. The samples were obtained
from the University Hospital, Federal University of Juiz de Fora,
Minas Gerais, Brazil, with the approval of the Ethics Committee for
Human Research (No. 098-22/2000). For this study, the controls
comprised five normal individuals and five patients with Chagas’
disease or leishmaniasis.
Paracoccidioides brasiliensis antigenic preparation.
Whole P. brasiliensis soluble antigenic preparation (WAg) was
obtained as described previously10. The isolate of P. brasiliensis
came from an individual with the acute form of PCM and the
inoculum was cultured for 15 days in Sabouraud medium. Fungal
suspensions were washed (3X) in sterile saline at 3,000 rpm for
5 min. The fungal mass was resuspended in an equal volume of
sterile saline containing phenylmethylsulfonyl fluoride (PMSF)
(20 µl/ml) and then subjected to sonication (100 cycles/3 min;
20X) in an ice bath. After centrifugation at 10,000 rpm for 40
min, the supernatant was dialyzed against distilled water for 48
h and stored at -20 ºC.
Periodate oxidation. Carbohydrate groups of WAg were
disrupted by treatment with sodium metaperiodate (Sigma),
which yielded a purified protein antigenic preparation (MPAg).
WAg was adsorbed onto flat-bottom Immunolon II microtiter
plates, washed twice with 0.05 M sodium acetate (pH 4.5) and
treated with sodium metaperiodate (10 mM in the acetate buffer)
for one hour in darkness at room temperature. Afterwards, the
plates were washed twice with sodium acetate and the reaction
was stopped by adding sodium borohydride (50 mM in PBS)
(Sigma) for 30 min at room temperature. The plates were then
washed twice with PBS.
Isotyping. Antibody isotype analyses were performed by
specific ELISA to detect anti- P. brasiliensis reactivity, using plates
adsorbed with WAg and MPAg P. brasiliensis antigens. Horseradish
peroxidase (HRP)-conjugated, isotype-specific mouse anti-human
IgG (anti-total IgG), anti-IgM, anti-IgA, anti-IgG1 or anti-IgG2
(Southern Biotechnology Associates, Inc.) were used and the
assays were developed by adding tetramethylbenzidine (TMB)
peroxidase substrate solution (Kirkegaard & Perry Laboratories).
The reaction was stopped by adding 1 M H2SO4, and the color
reaction was read at 450 nm in a microplate reader (Molecular
Devices Corp.).
Western blotting. WAg antigen was diluted in an SDS
sample buffer (2% v/v SDS, 2% v/v 2-ME, 10% v/v glycerol and
0.01% bromophenol blue in 0.125 M Tris-HCl buffer, pH 6.8),
heated at 95ºC for 5 min and analyzed by means of SDS-PAGE
on 10% polyacrylamide gels. After electrophoresis, the antigenic
fractions were transferred to nitrocellulose. The blots were
blocked with 0.05% PBS, Tween 20 and 5% nonfat dried milk
for 30 min. Serum samples (1:100) were added to each strip
for 60 min at room temperature and then they were washed
with blocking buffer. HRP-conjugated isotype-specific mouse
anti-human anti-IgG1 (1:1,000) was added to each strip. After
60 min, the strips were washed and the color reaction was
developed with 0.06% 4-chloro-1-naphthol in tris-buffered
saline (TBS) solution for 10 min. The reaction was stopped by
adding 3.3 µl of H2O2.
Statistical analysis. The Mann-Whitney U test was used to
analyze the positive significance (P < 0.05) of the immunoglobulin
levels in the serum from the two groups of patients. A paired
nonparametric test (Wilcoxon) was used to evaluate the
differences in the patients’ serum reactivity towards WAg and MPAg
antigenic preparations.
RESULTS
Serum immunoglobulin isotype reactivity in
paracoccidioidomycosis patients. Serum from patients with
both the acute and the chronic form of PCM showed higher reactivity
towards all immunoglobulin isotypes (total IgG, IgM, IgA, IgG1 and
IgG2) when WAg was used than when MPAg was used (P < 0.05)
(Figures 1 and 2). Serum samples from patients with the acute form
of PCM presented higher levels of total IgG (Figure 1A) and IgM
(Figure 1B) anti-P. brasiliensis antibodies than did serum samples
from patients with the chronic form of the disease, when the MPAg
was used (P < 0.05). On the other hand, IgA levels specific to WAg
were higher in the serum from chronic PCM patients than in the
serum from acute patients (Figure 1C) (P < 0.05).
There were no significant differences in the reactivity of serum
IgG1 and IgG2 between patients with the acute and chronic forms
of PCM, regardless of the antigenic preparation used (Figure 2).
However, the reactivity of the IgG1 (Figure 2A) and IgG2 (Figure
2B) isotypes in patients with PCM was higher (P < 0.05) than in
Revista da Sociedade Brasileira de Medicina Tropical 41(4):, jul-ago, 2008
the healthy controls or non-PCM diseased controls (Figure 2).
Serum from patients with chronic PCM showed only low levels
of specific IgG3 and IgG4 when either WAg or MPAg were used
(data not shown).
Serum reactivity distinguished by western blotting.
Western blot analysis with P. brasiliensis WAg using anti-human IgG1
polyclonal antibodies showed that antigens with molecular weights of
approximately 19 kDa, 27 kDa and 31 kDa were markedly recognized
by the serum immunoglobulins from patients with the acute form of
PCM. In contrast, serum from both the chronic and the acute PCM
patients reacted with antigens of 43 kDa and 75 kDa (Figure 3).
All serum samples were tested against whole antigen (WAg) ( ) or sodium
metaperiodate-treated WAg (MPAg) (□) of Paracoccidioides brasiliensis; horizontal
line (─) represents the median values of samples detected by ELISA. The optical density
(OD) was measured at 450 nm. * p < 0.05 vs. HC and Leish/CD groups
All serum samples were tested against whole (WAg) () or sodium metaperiodatetreated WAg (MPAg) (□) of P. brasiliensis; horizontal line (─) represents the median
values of samples detected by ELISA. The optical density (OD) was measured at 450
nm. * p < 0.05 vs. chronic patients (A and B), * p < 0.05 vs. acute patients (C).
Figure 1 - Reactivity of total IgG (A), IgM (B) and IgA (C) in serum
from patients with acute and chronic paracoccidioidomycosis. Serum
from PCM patients, healthy controls (HC) and diseased controls
(Leish = Leishmaniasis or CD = Chagas disease) were screened.
Figure 3 - Recognition profile of isotype-specific IgG1 from serum of PCM patients with the acute
form (A, lanes 1 - 10) and chronic form (B, lanes 1- 9), towards fractionated antigenic components
of Paracoccidioides brasiliensis (WAg) by western blot assay.
Figure 2 - Reactivity of IgG1 (A) and IgG2 (B) in the serum from
PCM patients, healthy controls (HC) and diseased controls (Leish =
Leishmaniasis or CD = Chagas disease).
Ferreira AP et al
DISCUSSION
Previous studies have shown the profile of serum
immunoglobulin isotypes that are reactive towards P. brasiliensis
antigens in acute and chronic forms of PCM, using P. brasiliensis
filtrate antigens or purified recombinant proteins1 11. To our
knowledge, this is the first study of serum reactivity in PCM that has
compared antigenic preparations obtained from P. brasiliensis
culture filtrates that differed according to their carbohydrate
content.
The use of serological tests for PCM diagnosis has been very
limited, mainly due to the high frequency of cross-reactivity with
other infections, such as American trypanosomiasis, leishmaniasis,
histoplasmosis and Jorge Lobo’s disease, among others18 19. In
order to improve the specificity, a purified 43 kDa glycoprotein
(gp43) was used in serological tests and showed high-specificity
results17 20. An additional study to evaluate the reactivity of gp43
in serological tests was performed after treatment of gp43 with
metaperiodate to disrupt the carbohydrates in the molecule,
and this showed a reduction in nonspecific reactivity18. Despite
these findings, no study with disruption of carbohydrates from
an unpurified antigenic preparation of P. brasiliensis has been
performed. In the present study, antigens obtained from P.
brasiliensis culture filtrate (WAg) were treated with metaperiodate
to obtain a carbohydrate-modified antigenic preparation (MPAg).
WAg and MPAg were used to compare differences in the serum
immunoglobulin isotype profile (total IgG, IgM, IgA, IgG1 and
IgG2) in groups of patients with the acute and chronic forms of
PCM, by means of ELISA.
The results presented here showed that serum from patients
with either acute or chronic PCM had higher levels of total IgG
and IgM reactivity towards WAg than against MPAg. However, only
the IgG anti-P. brasiliensis isotypes (and not the IgM isotypes)
were significantly increased in PCM patients, in comparison with
the controls. Furthermore, acute PCM patients showed increased
serum IgM and IgG levels only for MPAg. Several studies have
demonstrated increased IgM production in early P. brasiliensis
infection2 3 6. IgG is now seen to be higher in acute PCM patients
than in chronic PCM patients and the present study is in agreement
with other studies that showed that juvenile PCM patients had
higher IgG anti-gp43 levels than did patients with the adult form
of the mycosis1 6.
Analysis of IgA production also showed differences between
acute and chronic PCM patients. In contrast to the higher IgG
and IgM isotype reactivity in acute patients, there was higher IgA
reactivity in chronic patients, which was mainly anti-WAg antigenic
preparation. Together, these and previous observations7 suggest
that carbohydrate epitope removal leads to decreased levels of
IgA. On the other hand, higher levels of IgA reactivity towards the
whole antigen could indicate lack of specificity for the reaction21.
In addition, the increased levels of IgA in the chronic PCM patients
might relate to chronic mucosal antigenic stimulation, which is a
characteristic of the chronic form of PCM. It is very well known that
chronic inflammation of the mucosa stimulates production of IgA
antibodies1 9 10. In addition, the secretory IgA in saliva from PCM
patients may be indicative that it has a protective role regarding
antigen neutralization on mucosal surfaces15.
Previous studies showed that IgG1 antibodies were present,
frequently at high levels, in subacute and chronic PCM patients1 11.
Using both WAg and MPAg antigenic preparations, IgG1 and
IgG2 subclass levels were found to be similar in the two clinical
forms. However, a significant difference in IgG1 and IgG2 levels
was detected when comparing PCM patients with healthy controls
and non-PCM diseased controls. Isotype-specific IgG1 western
blot analysis of the reactivity towards fractionated P. brasiliensis
antigens indicated that acute patients consistently recognize a set of
antigens that are of approximately 19, 27 and 31 kDa, in contrast
with recognition of 43 and 75 kDa antigens, which was common
to both the acute and the chronic form. Similar findings were
described by another author16, showing that a 38 kDa antigen was
recognized more frequently and intensely by the serum of acute
PCM than by the serum of chronic patients. Another study showed
that components of the P. brasiliensis with 27, 34 and 43 kDa
were reactive towards IgG anti-P. brasiliensis antibodies13.
In conclusion, the present study suggests that the use of MPAg,
obtained from treatment of WAg with sodium metaperiodate, could
improve the serological test to distinguish between the chronic and
acute forms of PCM, by testing the reactivity towards IgG and IgM.
The measurement of total IgG and specific IgG1 or IgG2 was efficient
in distinguishing between PCM patients and control individuals,
using either WAg or MPAg. This study suggests that the detection of
MPAg-specific IgG-antibodies reacting with low molecular weight
antigens may be applied to the development of a better method for
immunodiagnosis of paracoccidioidomycosis.
ACKNOWLEDGEMENTS
We thank Daniel Stockdell for language assistance.
REFERENCES
1. Baida H, Bicelli PJ, Juvenale M, Del Negro GM, Mendes-Giannini MJ, Duarte AJ,
Benard G. Differential antibody isotype expression to the major Paracoccidioides
brasiliensis antigen in juvenile and adult form paracoccidioidomycosis. Microbes
and Infection 1:273-278, 1999.
2. Bernard G, Mendes-Gianini MJS, Juvenale M, Miranda ET, Duarte AJS.
Immunosuppression in Paracoccidioidomycosis: T cell Hyporesponsiveness
to two Paracoccidioides brasiliensis glycoproteins that elicit strong humoral
immune response. The Journal Infectious Disease 175:1263-1267, 1997.
3. Biagioni L, Souza MJ, Chamma LG, Mendes RP, Marques AS, Mota NGS, Franco M.
Serological of Paracoccidioidomycosis. II - Correlation between class-specific
antibodies and clinical forms of the disease. Transactions of the Royal Society of
Tropical Medicine Hygiene 78: 617-621, 1984.
4. Botteon FA, Camargo ZP, Bernard G, Coelho RF, Chamone DA, Itano EN.
Paracoccidioides brasiliensis - reactive antibodies in Brazilian blood donors.
Medical Mycology 40:387-391, 2002.
5. Brummer E, Castaneda E, Restrepo A. Paracoccidioidomycosis: in the update.
Clinical Microbiology Review 6:89-117, 1993.
6. Bueno JP, Mendes-Giannini MJ, Del Negro GM, Assis CM, Takiguti CK, ShikanaiYasuda MA. IgG, IgM and IgA antibody response for the diagnosis and follow-up
of paracoccidioidomycosis: comparison of counterimmunoelectrophoresis and
complement fixation. The Journal Medical Veterinary Mycology 35:213-217, 1997.
Revista da Sociedade Brasileira de Medicina Tropical 41(4):, jul-ago, 2008
7. Chen YL, Ben KL, Cao XM. The terminal monosaccharide of antigenic determinant
is involved in the binding of human sperm to specific monoclonal IgA antibody.
Shi Yan Sheng Wu Xue Bao 29:101-107, 1996.
15. Miura CS, Estevão D, Lopes JD, Itano EN. �����������������������������������
Levels of specific antigen (gp43),
specific antibody, and antigen-antibody complexes in saliva and serum of
paracoccidioidomycosis patients. Medical
����������������
Mycology 39: 423-428, 2001.
8. Del Negro GM, Garcia NM, Rodrigues EG, Cano ML, Aguiar MS, Lirio VdS, Lacaz
CS. The sensitivity, specificity and efficiency values of some serological tests used
in the diagnosis of paracoccidioidomycosis. Revista do Instituto de Medicina
Tropical de São Paulo 33:277-280, 1991.
16. Panunto-Castelo A, Freitas-da-Silva G, Bragheto IC, Nartinez R, Roque-Barreira
MC. Paracoccidioides brasiliensis exoantigens: recognition by IgG from patients
with different clinical forms of paracoccidioidomycosis. Microbes and Infection
5:1205-1211, 2003.
9. Fayette J, Dubois B, Vandenabeele S, Bridon JM, Vanbervliet B, Durant I,
Banchereau J, Caux C, Briere F. Human dentritic cells skew isotype switching of
CD-40-activated naive B cells towards IgA1 and IgA2. The Journal Experimental
Medicine 185:1909-1918, 1997.
17. Puccia R, Schenkman S, Gorin PA, Travassos LR. Exocellular
��������������������������
components of
Paracoccidioides brasiliensis identification of a specific antigen. Journal
Infection and Immunity 53:199-206, 1986.
10. Franco M, Bagagli E, Cunha M, Chamma LG, Fecchio D. Paracoccidioides
brasiliensis antigen batches from the same isolate show immunological and
biochemical differences. Mycopathology 135:13-19, 1996.
11. Juvenale M, Del Negro GMB, Duarte AJS, Benard G. Antibody isotypes to a
Paracoccidioides brasiliensis somatic antigen in sub-acute and chronic form
paracoccidioidomycosis. The
��������������������������������������������������
Journal Medical Microbiology 50:127-134, 2001.
12. Lacaz CS, Porto E, Martins JEC. Paracoccidioidomicose. In: Lacaz CS (ed)
Micologia Médica, 8th edição, Sarvier Editora, São Paulo, p. 248-261, 1991.
13. Martins R, Marquesa S, Alves M, Fecchio D, Franco MF. �������������������������
Serological follow-up of
patients with itraconazole using Dot-blot, ELISA and e Western-blot. Revista
�����������
do
Instituto de Medicina Tropical de São Paulo 39:261-269, 1997.
14. Mendes-Giannini MJ, Del Negro GB, Siqueira AM. Serodiagnosis. In: Franco MI,
Lacaz CS, Restrepo A e Del Negro GB (ed) Paracoccidioidomycosis, CRC Press,
Boca Raton, p. 345-363, 1994.
18. Puccia R, Travassos LR. 43-kilodalton glycoprotein from Paracoccidioides
brasiliensis: immunochemical reactions with sera from patients with
paracoccidioidomycosis, histoplasmosis, or Jorge Lobo’s disease. Journal Clinical
Microbiology 29:1610-1615, 1991.
19. Suzuki E, Toledo MS, Takahashi HK, Straus AH. A monoclonal antibody direct
to terminal residue of B-galactofuranose of a glycolipid antigen isolated from
Paracoccidioides brasiliensis: cross-reactivity with Leishmania major and
Trypanosoma cruzi. Glicobiology 7:463-468, 1997.
20. Taborda CP, Camargo, ZP. Diagnosis of paracoccidioidomycosis by dot
immnobindig assay for antibody detection using the purified and specific antigen
GP43. Journal Clinical Microbiology 32:554-556, 1994.
21. Yokota S, Amano K, Hayashi S, Kubota T, Fujii N, Yokochi T. Human antibody
response to Helicobacter pylori lipopolysaccharide: presence of an
immunodominant epitope in the polysaccharide chain of lipopolysaccharide.
Journal Infection and Immunity 66:3006-3011, 1998.